CN115058409B

CN115058409B - Triose phosphate isomerase as French phoenix tree pollen allergen and application thereof

Info

Publication number: CN115058409B
Application number: CN202210651544.9A
Authority: CN
Inventors: 魏继福; 许志强; 焦永欣; 宋乐彬
Original assignee: Jiangsu Cancer Hospital; Jiangsu Province Hospital
Current assignee: Jiangsu Cancer Hospital; Jiangsu Province Hospital
Priority date: 2022-06-09
Filing date: 2022-06-09
Publication date: 2023-10-27
Anticipated expiration: 2042-06-09
Also published as: CN115058409A

Abstract

The invention discloses a novel allergen triose phosphate isomerase of French karaya pollen and application thereof as a medicament for preventing, diagnosing and treating allergic diseases. Comprising the following steps: (a) a protein comprising the amino acid sequence shown in sequence 2; (b) An amino acid sequence comprising any amino acid substituted, modified, deleted or added in the amino acid sequence shown in the sequence 2, and a protein having an allergen activity or a hypoallergen activity comprising the amino acid sequence; (c) Comprises an amino acid sequence having an identity of 90% or more to the amino acid sequence shown in sequence 2, and a protein having an allergen activity comprising the same, or an allergen having an immunological cross-reactivity with the amino acid sequence shown in sequence 2. The allergen disclosed by the invention is discovered in the karaya pollen for the first time, and can be used for preparing a karaya pollen allergen detection kit or developing and applying an individual desensitization vaccine.

Description

Triose phosphate isomerase as French phoenix tree pollen allergen and application thereof

Technical Field

The invention belongs to the fields of molecular biology, immunology and bioinformatics, and particularly relates to triose phosphate isomerase serving as a French phoenix tree pollen allergen and application thereof.

Background

Allergic diseases, also known as allergic diseases, are a common chronic inflammatory disease. According to statistics, 30% -40% of people worldwide are afflicted with allergic problems, and allergic diseases become the sixth disease worldwide. Among them, allergic diseases mediated by IgE are most common, and include asthma, rhinitis, severe allergic reactions, drug allergies, food allergies, eczema, urticaria, angioedema, and the like. Pollen is one of the most important aeroallergens, pollinosis is an IgE-mediated allergic disease affecting the respiratory tract, skin and other systems caused by pollen allergens, and the incidence rate is rising year by year, especially in industrial areas.

French phoenix tree is widely planted as an ornamental tree in western Europe, north America, etc., and many cities in China such as Shanghai, nanjing, wuhan, etc. are also seen everywhere as a street tree. The pollen is short and dense in pollination period, and in recent years, the pollen concentration is remarkably increased, and the sensitization rate varies from 2% to 56% all over the world, so that the pollen is considered to be one of the important causes of pollinosis. During the last 10 years, a great deal of research has been conducted on the pollen of phoenix tree in france, especially in western european cities, and four allergens, pla a 1, pla a 2, pla a 3 and Pla a profilin, respectively, have been identified from the extract, but new allergens remain.

Currently, diagnosis and immunotherapy of allergens is still mainly based on crude extracts of allergens, which are complex mixtures of ingredients. Therefore, the identification and isolation of IgE-mediated allergens is a necessary task to improve the diagnosis and treatment of such increasingly clinical diseases. The discovery of new sensitization components is undoubtedly rich in the component information of the karaya pollen allergen, is favorable for the diagnosis and the treatment of the karaya pollen allergy and the individuation, and is expected to become a treatment scheme for replacing the pollen crude extract with clear components and clear action mechanism along with the development of scientific technology and the continuous deep research of the components of the karaya pollen allergen, thereby effectively preventing and treating the karaya allergic diseases.

Disclosure of Invention

The invention aims to: aiming at the defects existing in the prior art, the technical problem to be solved by the invention is to provide a novel allergen protein triose phosphate isomerase of French karaya pollen.

The invention also solves the technical problems of providing the purification preparation of the triose phosphate isomerase of the French karaya pollen, the cloning of nucleic acid or gene sequences, the coding of the triose phosphate isomerase of the French karaya pollen allergen and the research application of the sensitization thereof.

The invention also solves the technical problem of providing an expression cassette, a recombinant vector, a recombinant protein, a recombinant cell or a recombinant bacterium.

The invention also solves the technical problem that the application of the French karaya pollen allergen, the nucleic acid or gene, the expression cassette, the recombinant vector, the recombinant protein, the recombinant cell or the recombinant strain in preparing a kit for diagnosing the karaya pollen allergic diseases or a medicament for preventing or treating the karaya pollen allergic diseases is passed through.

The invention finally solves the technical problem of providing a medicine or vaccine for preventing or treating the allergy of the phoenix tree pollen or a kit for diagnosing the allergen.

The technical scheme is as follows: in order to solve the technical problems, the invention provides an allergen protein triose phosphate isomerase of French karaya pollen, which comprises the following components:

(a) As set forth in SEQ ID NO:2, and a protein consisting of an amino acid sequence shown in the formula 2; or (b)

(b) A protein having an allergen activity or a hypoallergenic activity, wherein the amino acid sequence in (a) is substituted, modified, deleted or added with one or more amino acids; or (b)

(c) A protein having an allergen activity, which comprises an amino acid sequence having an identity of 90% or more to the amino acid sequence of (a); or (b)

(d) An allergen protein having immunological cross-reactivity with the protein of (a).

The present invention also includes amino acids comprising the allergen protein triose phosphate isomerase, said amino acids comprising:

(2a) As set forth in SEQ ID NO:2, and a polypeptide sequence represented by the following formula (2);

(2b) At least 90% homology with the amino acid sequence shown in (2 a);

(2c) And (2) complementary sequence of the amino acid sequence shown in (2 a), and an active allergen protein hybridized with the complementary sequence.

The present disclosure also includes a nucleic acid or gene encoding the allergen protein triose phosphate isomerase or the amino acid, the nucleic acid or gene comprising:

(3a) As set forth in SEQ ID NO:1, and a nucleotide sequence shown in the specification;

(3b) At least 90% homology with the nucleotide sequence of (3 a);

(3c) A single stranded DNA or RNA sequence complementary to the nucleotide sequence of (3 a).

The invention also includes an expression cassette, a recombinant vector, a recombinant protein, a recombinant cell or a recombinant bacterium comprising the nucleic acid or gene.

The invention also comprises the application of the French karaya pollen allergen triose phosphate isomerase, the nucleic acid or gene, the expression cassette, the recombinant vector, the recombinant protein or the recombinant cell in preparing a kit for diagnosing the karaya pollen allergic diseases or a medicament for preventing or treating the karaya pollen allergic diseases.

The invention also comprises the application of the allergen triose phosphate isomerase of the French karaya pollen, the amino acid, the nucleic acid or gene, the expression cassette, the recombinant vector, the recombinant protein, the recombinant cell or the recombinant bacterium in preparing a medicament or a reagent for detecting the content of triose phosphate isomerase specific IgE in serum of a patient.

The invention also comprises any preparation for preventing, diagnosing and treating allergic diseases, which is related to the following (d) - (f):

(d) A prophylactic or therapeutic agent comprising the allergen protein triose phosphate isomerase, the amino acid or the nucleotide as an active ingredient;

(e) A prophylactic or therapeutic agent comprising at least one of an immunomodulatory T-cell-reactive polypeptide fragment, B-cell-reactive polypeptide fragment, and a protein or polynucleotide derived from said allergen protein triose phosphate isomerase fused to a carrier protein;

(f) A therapeutic antibody specific for said allergen protein, said antibody comprising one of a polyclonal antibody, a monoclonal antibody, a chimeric antibody or a humanized modified antibody.

The invention also comprises a medicine for preventing or treating allergic diseases, which is a single or compound preparation and comprises the allergen protein triose phosphate isomerase, the amino acid or the nucleic acid or the gene.

Wherein the drug is an allergen vaccine. The allergen vaccine comprises a DNA vaccine, a recombinant live bacteria vaccine and a recombinant protein vaccine.

The invention also includes a kit for allergen diagnosis, comprising the allergen protein triose phosphate isomerase, the amino acid, or the nucleic acid or gene.

Wherein the kit further comprises a solid phase carrier for adsorbing or linking the allergen.

The beneficial effects are that: the invention discloses a method for identifying a novel component with serum-specific IgE binding activity from karaya pollen allergen by excavating allergen components in karaya pollen and finally combining traditional chromatographic separation and purification and tandem mass spectrometry identification technology, wherein the novel component is a gene sequence and an amino acid sequence of triose phosphate isomerase of the allergen of karaya pollen in France. Through research on the allergen triose phosphate isomerase, the allergen triose phosphate isomerase can be used for desensitizing treatment and prevention of allergic diseases through preparing allergen vaccines or related medicaments. Therefore, the invention has important value for clinical diagnosis and treatment of patients suffering from allergy to phoenix tree pollen.

Drawings

FIG. 1A is an elution profile of a Firmiana pollen extract on a HiTrap Q HP anion exchange column, with the arrowed portions combined for further purification, and the black box containing the Firmiana pollen triose phosphate isomerase.

FIG. 1B is an elution pattern on a Superdex G75 inch chromatographic column after combination, wherein the 5-6 parts (black boxes) are parts containing triose phosphate isomerase from Firmiana straminea pollen.

FIG. 1C is a map of the combined fractions purified on a 1ml HiTrap SP HP column, with the black box-labeled portion being the purified triose phosphate isomerase protein from Firmiana straminea.

FIG. 2 shows 12 peptide fragments of triose phosphate isomerase protein of phoenix tree pollen obtained by Nano-LC MS/MS analysis.

FIG. 3 is an electrophoretogram of the amplified product of the sequence encoding triose phosphate isomerase of phoenix tree pollen, the leftmost M lane being DNA 2000 Maker; lanes 1-10 are PCR amplified products.

FIG. 4 is a cDNA sequence encoding triose phosphate isomerase from karaya pollen and the corresponding amino acid sequence, underlined and bolded are the peptides identified by LC-MS/MS.

FIG. 5 shows ELISA to verify the binding activity of triose phosphate isomerase with specific IgE in serum of patients with allergy to karaya pollen, and 24 patients with allergy to karaya pollen showed positive reaction in serum of 58 patients with allergy to karaya pollen.

Detailed Description

1. Pollen allergen proteins

The pollen allergen protein provided by the invention is a protein selected from the following (a) - (c).

(a) A protein comprising an amino acid sequence represented by sequence 2;

(b) A protein having an allergenic activity or a hypoallergenic activity, which comprises an amino acid sequence comprising an amino acid sequence in which any amino acid in the amino acid sequence shown in sequence 2 has been substituted, modified, deleted or added;

(c) Comprises a protein having an allergen activity comprising an amino acid sequence having an identity of 90% or more to the amino acid sequence shown in sequence 2, or an allergen protein having immunological cross-reactivity with a protein comprising an amino acid sequence shown in sequence 2.

2. Amino acids constituting pollen allergen proteins

The amino acid of the present invention is an amino acid sequence constituting the pollen allergen protein, and comprises amino acids of the allergens described in the following (a) to (c).

(a) Comprising the amino acid sequence shown in sequence 2;

(b) At least 90% homology with said amino acid sequence 2;

(c) A complementary sequence comprising the amino acid sequence shown in sequence 2, and an active allergen protein formed by hybridization of the complementary sequence;

3. nucleotide sequence encoding pollen allergen protein

The nucleotide of the invention is a nucleotide sequence for encoding the pollen allergen protein, and comprises the nucleotide sequences (a) - (c).

(a) Comprising the nucleotide sequence shown in sequence 1;

(b) At least 90% homology to said nucleotide sequence 1;

(c) Single-stranded DNA or RNA sequence complementary to said nucleotide sequence 1

4. Acquisition of polynucleotides encoding pollen allergen proteins

The polynucleotide encoding pollen protein of the present invention can be obtained by cloning, and the cloning method is not limited.

5. Pollen allergen protein production

The allergen protein of the invention is obtained by separating and purifying the extract of the phoenix tree pollen. The separation and purification method is not particularly limited.

The recombinant plasmid prepared by inserting the polynucleotide of the present invention into a vector may be introduced into a host cell, and the pollen allergen protein may be expressed and collected in the cell or outside the cell. The vector and host of the present invention are not particularly limited.

The vector into which the polynucleotide of the present invention is inserted is not particularly limited as long as it can replicate in the above-mentioned host, and may be appropriately determined depending on the type of host to be introduced, the method of introduction, and the like.

6. Preventive, diagnostic or therapeutic agent for allergic diseases

The pollen allergen protein of the present invention can be used for producing a prophylactic or therapeutic agent for allergic diseases including all allergic diseases caused by external specific antigens;

the preventive or therapeutic agent for allergic diseases according to the present invention comprises:

(a) The pollen allergen protein of the present invention is directly used or dried to be powder;

(b) The formulation may be formulated by adding various auxiliary materials such as stabilizers, excipients, dissolution aids, opacifiers, buffers, painless agents, preservatives, colorants and the like as required.

7. Antibodies to pollen allergen proteins

The antibody of the pollen allergen protein of the present invention is an antibody capable of specifically binding to the pollen allergen protein of the present invention. The antibody refers to immunoglobulin (IgA, igD, igE, igG, igM and Fab fragment, F (ab') 2 fragment, and Fc fragment thereof), and examples thereof include polyclonal antibody, monoclonal antibody, single chain antibody, anti-idiotype antibody (anti-idiotype antibody), and humanized antibody, but are not limited thereto.

The antibody may be produced by various known methods, and the production method is not particularly limited.

The antibody can be used for identification of organisms expressing the pollen allergen protein of the present invention, tissues or cells thereof, and the like.

The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

EXAMPLE 1 purification of the French Firmianae pollen allergen triose phosphate isomerase

Weighing 8.0. 8.0 g phoenix tree pollen to extract total protein: pollen was first mixed with 60 mL of 0.01M Phosphate Buffer (PBS), added with a final concentration of 1. 1 mM protease inhibitor (PMSF), incubated upside down at 4℃overnight, centrifuged at 12000 Xg, and the supernatant was collected and filtered through a 0.22 μm membrane for later use.

Purification of triose phosphate isomerase in phoenix tree pollen: the karaya pollen extract was isolated by anion exchange, gel filtration and cation exchange chromatography steps on the Ä KTA-go system (GE Healthcare, uppsala, sweden). Specifically, crude pollen extract was desalted with HiTrap desalting column equilibrated with 0.02M Tris-HCl, pH 8.4, and the collected fractions were adsorbed on HiTrap Q HP column pre-equilibrated with the same buffer as described above (Uppsala, sweden), and the adsorbed proteins were eluted with 0.02M Tris-HCl solution containing 1M sodium chloride in a gradient of 0% to 100%. Eluted fractions were stained by SDS-PAGE and Coomassie Brilliant blue R250, fractions with similar protein distribution were pooled and separated in Superdex G75, the column equilibrated with 50 mM phosphate buffer (pH 7.2) containing 150 mM sodium chloride, eluted at a flow rate of 0.5 mL/min, and the fractions obtained were analyzed by SDS-PAGE and pooled for further separation. The pooled fractions were further adsorbed onto a 1mL HiTrap S HP (Uppsala, sweden) column, eluted with 50 mM 2-morpholinoethanesulfonic acid, pH 6.0, flow rate 1mL/min, and analyzed by SDS-PAGE electrophoresis, and finally purified to migrate as a single band to about 27kDa on SDS-PAGE.

EXAMPLE 2 internal peptide fragment identification

Identification of IgE binding proteins by LC-MS/MS: the 27kDa protein purified in example 1 was first separated on SDS-PAGE and stained with Coomassie Brilliant blue R250. Protein bands were excised from the gel, digested with trypsin and analyzed by mass spectrometry, and raw data were analyzed using pFind software to obtain 12 internal peptide fragments with 89.3% coverage of the amino acid full length.

EXAMPLE 3 cloning of full-Length cDNA of triose phosphate isomerase from Firmiana pollen

The result of the mass spectrum is analyzed by pFInd software, and the internal peptide fragment is obtained. The peptide fragment was matched to the high throughput pollen transcriptome sequencing results to obtain the theoretical cDNA sequence encoding the triose phosphate isomerase of karaya pollen, and the sequence was further confirmed by T-A cloning and Sanger sequencing. Specifically, total RNA of karaya pollen is first extracted and reverse transcribed to obtain cDNAs. According to the cDNA sequence of the theoretical phoenix tree pollen triose phosphate isomerase obtained by matching, designing a primer for PCR amplification, wherein an upstream primer is 5'-CTCTAGCCTTACTGTGTT-3', a downstream primer is 5'-CCCATACCTGATTCCAAG-3', and carrying out PCR amplification of an actual sequence in a 25 mu L system: exTaq enzyme (0.125. Mu.l), 10 XExTaq buffer (2.5. Mu.l), dNTP mix (2. Mu.l), firmiana pollen cDNA product (1. Mu.l), upstream and downstream primer mix (1. Mu.l), sterile distilled water was supplemented to 25. Mu.l. The PCR amplification conditions were 98℃/5s (one cycle) for pre-denaturation, 98℃/10s for denaturation, 55℃/30s for annealing and 72℃/1min for extension (30 cycles), and 72℃/10min for re-extension (one cycle). The PCR amplified product was recovered, purified and ligated into pCE2 TA/Blunt-zero plasmid vector and transformed into JM109 competent cells. Positive clones were selected on Luria-Bertani (LB) plates containing 100. Mu.g/mL kanamycin, and confirmed by DNA sequencing to obtain the actual sequence of triose phosphate isomerase natural protein of phoenix tree pollen, the gene has the full length of 762 bp, codes 253 amino acids and has the theoretical molecular weight of 27kDa. The amino acid sequence is shown as SEQ ID NO. 2.

Further carrying out recombination reaction. Firstly, the plasmid of the clone of the triose phosphate isomerase of the phoenix tree pollen and the plasmid of the pET28a escherichia coli are extracted by using a ClonExpress II One Step Cloning Kit (Novozan biotechnology Co., nanj, china) kit (the operation steps refer to the instruction provided by the kit), then the nucleotide sequence of the coding region of the clone is connected between NcoI and XhoI sites of the pET28a+ plasmid through a recombination reaction to obtain a recombinant plasmid, the recombinant plasmid is transformed into BL21 (DE 3) host bacteria (Novozan biotechnology Co., nanj, china) through heat shock, the flat cloning is carried out, the single clone bacteria are picked up and cultured in a liquid culture medium at 37 ℃, and the successful construction of the plasmid is confirmed through sequencing.

Example 4 ELISA experiments demonstrated the sensitization of triose phosphate isomerase to Firmiana pollen

The triose phosphate isomerase of phoenix tree pollen prepared in example 1 was coated in a 96-well plate overnight. Then blocked with a solution of PBST (PBS containing 0.1% Tween 20) containing 1% Bovine Serum Albumin (BSA) at 37℃for 1.5h. After washing with PBST, 58 cases of serum from patients with allergy to karaya pollen (serum from people's hospitals of Jiangsu province) were added, and incubated at 37℃for 1.5. 1.5h. The serum of the non-allergic patient is used as a negative control. Incubate 1 h with 100. Mu.L of anti-human IgE-horseradish peroxidase conjugate (1:2500 dilution) at room temperature. After washing, the addition of the color-developing solution was stopped after 13 minutes, and the detection was performed by an ELISA reader OD 450. The results showed that 24 out of the serum of 58 patients with allergy to karaya pollen were positively reacted to triose phosphate isomerase, and the sensitization rate was 41% in the patients with allergy to karaya.

Sequence listing

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tgccacggtg aagaatgctt aagcttgggg cttggaatca ggtatggg 828

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Claims

1. An allergen protein triose phosphate isomerase of karaya pollen, characterized in that: the amino acid sequence of the allergen triose phosphate isomerase is shown in SEQ ID NO: 2.

2. A nucleic acid molecule encoding the allergen protein triose phosphate isomerase of claim 1, wherein the nucleic acid molecule has a nucleotide sequence set forth in SEQ ID NO: 1.

3. An expression cassette, recombinant vector, recombinant cell or recombinant bacterium comprising the nucleic acid molecule of claim 2.

4. Use of the triose phosphate isomerase, which is an allergen of karaya pollen, of claim 1, the nucleic acid molecule of claim 2, the expression cassette, recombinant vector, recombinant cell or recombinant bacterium of claim 3 in the manufacture of a kit for diagnosing a allergic disease of karaya pollen or a medicament for preventing or treating a allergic disease of karaya pollen or in the manufacture of a medicament or reagent for detecting the content of triose phosphate isomerase-specific IgE in serum of a patient.

5. Any formulation for preventing, diagnosing, treating allergic diseases related to: a prophylactic or therapeutic agent comprising the allergen protein triose phosphate isomerase of claim 1 or the nucleic acid molecule of claim 2 as an active ingredient.

6. A medicament for preventing or treating allergic diseases, characterized in that the medicament is a single or compound preparation comprising the allergen protein triose phosphate isomerase of claim 1 or the nucleic acid molecule of claim 2.

7. The medicament of claim 6, wherein the medicament is an allergen vaccine.

8. A kit for allergen diagnosis, characterized in that it comprises the allergen protein triose phosphate isomerase of claim 1 or the nucleic acid molecule of claim 2.

9. The kit of claim 8, further comprising a solid support that adsorbs or binds the allergen protein triose phosphate isomerase of claim 1.